Real time DNA amplification and detection methods are efficient for sequence identification and quantification of a target since no pre-hybridization amplification is required. Amplification and hybridization are combined in a single step and can be performed in a fully automated, large-scale, closed-tube format.
Methods that use hybridization-triggered fluorescent probes for real time PCR are based either on a quench-release fluorescence of a probe digested by DNA Polymerase (e.g., methods using TaqMan, MGB-TaqMan) or on a hybridization-triggered fluorescence of intact probes (e.g., molecular beacons,.and linear probes, see U.S. Pat. Nos. 6,030,787, and 5,723,591). In general, the probes are designed to hybridize to an internal region of a PCR product during annealing stage. For those methods utilizing TaqMan and MGB-TaqMan the 5′-exonuclease activity of the approaching DNA Polymerase cleaves a probe between fluorophore and quencher thus releasing fluorescence.
What is needed in the art are new oligonucleotide probes and conjugates that are useful in “real-time” amplification processes and which can be prepared with a variety of nucleotide bases in short lengths. The present invention provides such probes and conjugates, as well as methods for their use.